There is a great clinical need for superior heart valve replacements for patients of all ages. Current valve substitutes provide excellent quality of life but have limited durability, mostly because these are non-living implants. The most physiologic valve replacements are considered the valve homografts, which have excellent hemodynamics, but lack cells. Therefore, the ideal valve substitute would be an in vitro regenerated, living construct which closely mimics the unique biological and hemodynamic features of the aortic root. The aortic root comprises distinct anatomical components, extracellular matrix molecules and cells, which maintain a balanced matrix homeostasis; the secret to life-long mechanical endurance. We hypothesized that functional aortic root regeneration in vitro is possible by seeding each anatomical component of xenogenic acellular roots with adult stem cells or pre-differentiated adult stem cells, and exposing cell-seeded roots to controlled mechanical cues to induce stem cell differentiation and maturation into target cells as a response to the niche biology and biomechanics. Proposed aims systematically approach testing this hypothesis by accomplishing internal and external seeding of acellular roots with cells, followed by progressive exposure to mechanical stimuli adaptation and dynamic conditioning, reaching aortic valve hemodynamic conditions. Pilot studies demonstrate feasibility of each proposed technique, including development of novel microarray-based seeding approaches, 3D rotators for adaptation and advanced valve bioreactors for conditioning. A multi-PI team of world renowned experts in each of the critical fields has been put together to ensure successful completion of the study. It is expected that such procedures will lead to mechanically robust, fully functional, viable aortic valve roots populated with metabolically active cells capable of matrix remodeling. This would ensure hemodynamic functionality immediately after implantation as well as long- term mechanical and biological stability due to continuous cell-mediated matrix repair. These replacement valves, developed with the aid of technologically advanced, integrated regeneration platforms would be ready for preclinical translational testing in large animals.